Biological product and process of obtaining same



Patented Nov. 10, 1942 BIOLOGICAL PRDDUUI AND PROCESS OF OBTAINING SAMENorbert H. Fell, Detroit, Micln, assignor to Parke,

Davis & Company, Detroit, Micln, a corporation of Michigan N 0 Drawing.

Application August 18, 1938,

Serial No. 225,612

8 Claims.

The invention relates to preparation of products useful in preventingthe allergic symptoms to which many people are subject as the result oftheir sensitivity to specific sensitizing substances. The inventionrelates more particularly to an antigenic product which can beartificially or synthetically produced and which is useful fordeveloping either active or passive immunity in sensitized individuals.

It has been shown that histamine or related substances can be found inthe blood of animals during'anaphylactic shock. I have now found that itis possible to produce both active and passive immunity againsthistamine and similar substances produced during manifestation of thesymptoms of anaphylaxis and other allergic reactions whereby it ispossible for sensitive individuals to avoid allergic reactions,regardless of the type of allergens to which they are sensitive.

In carrying out the invention I combine histamine, or like substanceproduced in the body during an allergic reaction, with a large moleculeor particle in a haptenic or haptene-like linkage to form a complexwhich can be used as an antigen for developing active immunity in thebody of a sensitized individual, or for injecting into animals fromwhich an antiserum can thereafter be obtained containing an antibody forneutralization of the effects of histamine or histamine-like substances.

My invention makes possible a type of treatment of allergic conditionswhich is non-specific and does not depend upon recognition andprotection against the individual allergens which may produce symptomsin any given case.

The preferred method of carrying 'out the invention is to prepare adiazotized derivative of histamine itself and couple .the latter with aprotein molecule. This may be done in various ways. For example, theamino group of histamine can be combined through an amide linkage withan aliphatic or aromatic carboxylic acid containing an amino group or anitro group capable of reduction to an amino group, and the amino amidethereby formed diazotized at the free amino substituent and. theresulting diazo compound coupled with a protein to give ahistamineazo-protein complex useful in producing active and passiveimmunity to histamine and like substances responsible for themanifestations of allergy.

The invention in its broader aspects is described and claimed in mycopending application, Serial No. 371,456, filed December 23, 1940. The

, such species.

The invention may be illustrated by the following examples.

EXAMPLE l.Preparation of (1. nitro benzamide.

intermediate Two moles of histamine base are dissolved in hot drychloroform and a solution of one mole of p-nitro benzoyl chloridedissolved in ether is stirred in. A heavy precipitate forms immediatelywhich has a waxy appearance. The supernatant liquid layer is removedfrom the precipitate and centrifuged to remove all insoluble material.The precipitate is dissolved in boiling water, filtered while hot fromany material which falls to dissolve, and the filtrate cooled to givecrystals of the imidazolyl ethyl p-nitro benzamide. The crystals can bewashed with acetone and ether and then dried at 110 C. They melt at 204C. The compound prepared by this example can be represented by theformula,

HN N I H An analysis of the crystalline product gives the theoreticalamount of nitrogen for a compound of instant application is directedmore particularly 55- compound One-half gram of imidazolyl ethyl p-nitrobenzamide, prepared as in Example 1, is added to 50 cc. of a hotsolution of 3.5 grams of ferrous sulfate (FeSOdHzO). 35 cc. of a 2.5%solution of ammonium hydroxide are then gradually added to the ferroussulfate solution while heating on the steam bath. After adding all theammonium hydroxide solution, the reaction mixture is heated for 30minutes and the ferric hydroxide filtered off and the filtrate set asideto cool. The filtrate contains the imidazolyi ethyl p-amino benzamide.This compound may be obtained in solid form by allowing the filtrate tostand whereupon spontaneous crystallization sometimes occurs, or,evaporating ofi the solvent, or adding an organic solvent in which theamino benzamide is insoluble, thereby obtaining a precipitate orcrystals which can be filtered off.

The crystals have a melting-point of 180-182 C. and differ from thecorresponding p-nitro compound in being very soluble in acid and veryinsoluble in alkali. The product of this example is represented by theformula,

EXAMPLE 3.Preparatzon of dz'azo compound of z'midaaolyl ethyl p-aminobenzamz'de The filtrate from Example 2 containing the imidazolyl ethylp-amino benzamide is neutralized with hydrochloric acid and 173 mgms. ofsodium nitrite added while chilling the reaction mixture in an ice-saltmixture. The reaction mixture is acidified with 4.5 cc. of normal H01and allowed to stand for about 20 minutes. It contains the diazotizedimidazolyl ethyl p-amino benzamide.

EXAMPLE 4.--Preparation of a histamine-a20- protein antigen Five gramsof casein are taken up in 50 cc. of cold water and 50 cc. of boilingwater added. The solution is then adjusted to a pH of 8.1-8.2: withsodium hydroxide solution. This alkaline casein solution is chilled inan ice-bath. A solution of a diazonium salt of imidazolyl ethyl p-aminobenzamide, prepared for instance as described in Examples 1, 2 and 3 andstarting with about 830 mgms. of histamine base, is added slowly to thechilled casein solution. More 10% NaGI-I is added until the mixtureturns a bloodred color. Any insoluble material present at this stage canbe centrifuged off and discarded. An excess of hydrochloric acid isadded to the supernatant liquid until it becomes acid to Congo redpaper. This acidification causes a heavy flocculent precipitate to comeout. The precipitate is centrifuged off. The centrifuged precipitate iswashed with acidified saline and then dissolved in excess dilute alkalihydroxide to give a clear blood-red solution. To this solution, 0.5%phenol is added and then a few drops of concentrated HCl to bring the pHto about 7.6, after which the solution is put through a Mandler filter.The solution is ampouled and can be submitted to sterility tests beforeusing it for injections.

This antigen injected into animals sensitized to horse serum confers onthem protection against subsequent anaphylactic shock from horse serum.Animals can be immunized with this histamineazo-protein antigen; theycan be bled subsequently, and their blood serum can be shown to containantibodies against the complex antigen and against histamine. Such animmune serum injected into guinea-pigs sensitized to horse serumprovides them with protection (passive immunity) against subsequentanaphylactic shock from horse serum.

Human subjects may also be treated in the same manner as animals to givethem an active or a passive immunity which enables them to avoid theuncomfortable and deleterious symptoms accompanying allergic reactions.

Instead of using casein in this example, I may use some other protein,such as egg albumin or large organic molecules capable of coupling witha histamine-diazo compound or like combination of histamine orhistamine-like compound with a diazotized amino compound.

Instead of using nitro benzoyl chloride. other nitro organic acidhalides may be used to combine with the histamine to form an amidecombination capable of reduction to give an free amino group forpurposes of diazotization.

The invention is not limited to any particular method of producing thehistamine amide intermediate. The essential feature of the preferredform of the invention is to produce a derivative of histamine containingan amino group, other than the amino group of histamine, which can bediazotized to give a diazonium salt for purposes of coupling with theprotein or like organic molecule.

Although I prefer to make a histamine-a20- protein complex as an antigenfor the development of immunity against the deleterious products, suchas histamine itself, produced in the human body during an allergicreaction, the invention is not limited to use of such combinations ofhistamine and similar compounds. It also embodies various loosecombinations of histamine, either chemical or physical in nature, orboth, with a larger molecule or particle. For example, histamine may beabsorbed on kaolin or other colloidal organic or inorganic substance ina combination which is probably more physical than chemical in nature,and this absorbed combination-can be injected into animals which, aftera time, will yield an antiserum to histamine itself.

In the claims appended hereto the expression animal body is intended toinclude the human body as well as the bodies of animals.

What I claim as my invention is:

1. Method of producing immunity against histamine which comprisescombining histamine with an amino-substituted carboxylic acid by way ofthe amino group of histamine to form an amino amide of histamine,diazotizing said amino amide, chemically coupling the diazotizedcompound With a protein of the class consisting of casein and egg whiteto form an antigenic complex, and administering said complex to ananimal body for the development therein of an active immunity againsthistamine.

2. Method of producing immunity against histamine which comprisescombining histamine with a p-nitro benzoyl halide to form an imidazolylethyl p-nitro benzamide, reducing the nitro group of the latter to anamino group with production of the corresponding amino amide,diazotizing said amino amide, chemically coupling the diazotized aminoamide with a protein of the class consisting of casein and egg white toform an antigenic complex, and administering said complex to an animalbody for the development therein of an active immunity againsthistamine.

3. Method of producing immunity against histamine which comprisescombining histamine with p-nitro benzoyl chloride to form imidazolylethyl p-nitro benzamide, reducing the nitro group of the latter to anamino group with production of imidazolyl ethyl p-amino benzamide,diazotizing said amino amide, chemically coupling the diazotized aminoamide with caseing to form an antigenic complex, and administering saidcomplex to an animal body for the development therein of an activeimmunity against histamine.

i. The product of chemically coupling a protein of the class consistingof casein and egg white with a diazotized amino-substituted carboxylicacid amide of histamine.

5. The product of chemically coupling a protein of the class consistingof casein and egg white with a diazotized imidazolyl ethyl p-aminobenzamide.

6. The product of chemically coupling casein with diazotized imidazolylethyl p-amino benzamide.

7. Method of producing immunity against histamine which comprisescombining histamine with an amino-substituted carboxylic acid by way ofthe amino group of histamine to form an amino amide of histamine,diazotizing said amino amide, chemically coupling the diazotizedcempound with casein to form an antigenic complex, and administeringsaid complex to an animal body for the development therein of an activeimmunity against histamine.

8 The product of chemically coupling casein with a diaziotizedamino-substituted carboxylic acid amide of histamine.

NORBERT H. FELL,

